Regulation of zinc deficiency and tolerance in plants

ABSTRACT

The invention relates to a method to change the capacity of a plant for adaptation to changes in the zinc concentration in the environment, especially the soil, comprising providing said plant with a nucleotide according to SEQ ID NO:1 or SEQ ID NO: 2 or an ortholog thereof. Such a method is especially useful to decrease Zn deficiency or to (hyper)accumulate Zn in plants for bioremediation or for biofortification. Transgenic plants for these proteins are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a divisional of application Ser. No. 13/383,808(allowed) having an international filing date of 16 Jul. 2010, which isthe national phase of PCT application PCT/NL2010/050461 having aninternational filing date of 16 Jul. 2010, which claims benefit ofEuropean patent application No. 09165714.8 filed 16 Jul. 2009. Thecontents of the above patent applications are incorporated by referenceherein in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

The entire content of the following electronic submission of thesequence listing via the USPTO EFS-WEB server, as authorized and setforth in MPEP § 1730 II.B.2(a)(C), is incorporated herein by referencein its entirety for all purposes. The sequence listing is identified onthe electronically filed text file as follows:

File Name Date of Creation Size (bytes) 313632012910SeqList Oct. 18,2018 28,751

FIELD OF THE INVENTION

The invention relates to plant biotechnology, more specifically tomodulate the sensitivity to metals in plants, more specifically to Zn.Particularly, the invention relates to adaptation of the plant tochanges in the availability of Zn in the environment.

BACKGROUND OF THE INVENTION

Zinc is an essential micronutrient for all living organisms includingplants. Zinc is typically the second most abundant transition metal inorganisms after iron (Fe), and the only metal represented in all sixenzyme classes (oxidoreductases, transferases, hydrolases, lyases,isomerases and ligases). Zinc binding sites also occur in a wide rangeof other proteins, membrane lipids and DNA/RNA molecules. The largestclass of Zn-binding proteins in organisms is the zinc finger domaincontaining proteins that function as transcription regulator.

Zn is present in the soil in primarily three fractions: (i)water-soluble Zn (including Zn²⁺ and soluble organic fractions), (ii)adsorbed and exchangeable Zn in the colloidal fraction (associated withclay particles) and (iii) insoluble Zn complexes and minerals. Zinc isacquired from the soil solution primarily as Zn²⁺, but also potentiallycomplexed with organic ligands, by roots which feed their shoots via thexylem.

When facing a shortage in zinc supply, plants adapt by enhancing thezinc uptake capacity. Plants are thought to control Zn homeostasis usinga tightly regulated network of zinc status sensors and signaltransducers controlling the coordinated expression of Zn transportersinvolved in Zn acquisition from the soil, mobilization between organsand tissues and sequestration within cellular components (Clemens, S.,2001, Planta 212:475-486).

In recent years numerous studies have been performed to unravel thebiochemical pathways of Zn uptake and transport. In these studies,however, it is not yet found which proteins are responsible for Znuptake from the soil (Palmer, C. M. and Guerinot, M. L., 2009, NatureChem. Biol. 5:333-340). While candidate genes for the required Zntransporters have been identified, the so-called ZIP transporters ZIP1,ZIP2, ZIP3 and ZIP4 (Grotz, N. et al., 1998, Proc. Natl. Acad. Sci USA95:7220-7224), more proteins seem to be necessary to explain thephenomenon of Zn-hyperaccumulation, such as the heavy metal transportersHMA2, 3 and 4 (Hanikenne et al, 2008, Nature 453:391-395). Alsonicotianamine, made by nicotianamine synthase (NAS), seems to play arole in vascular transport of zinc, and the citrate transporter FRD3(Durrett, T. P. et al., 2007, Plant Physiol. 144:197-205). For transportbetween tissues and organs several proteins seem to be involved. Membersof the YSL group, a subfamily of the oligopeptide transporter (OPT)family of trasnporters, are proteins that have been mentioned. Forintracellular transport of Zn, NRAMP ZIP and ZIF proteins seem to beinvolved. Transport of Zn into the vacuole is performed by MTPs (metaltolerance proteins, also referred to as cation diffuserfacilitator—CDF-proteins).

(Lack of) adaptation of plants to a changing concentration of Zn in theenvironment works both ways: zinc deficiency and zinc toxicity. After Fedeficiency, Zn deficiency is the most commonly occurring micronutrientdeficiency in agriculture, mainly affecting parts of Asia (Turkey andNear-East Asia, Central Asia, South and Central China), Sahel andsub-Saharan Africa and Australia. Since plants are often the majordietary source of micronutrients for human consumption, many peopleworldwide suffer from Fe and Zn deficiencies as plants, especiallycereals, are notoriously poor in their content of bioavailable Fe andZn.

Some soils are not deficient in minerals, but have become contaminatedwith large amounts of heavy metals, such as Zn or Cd. Zn toxicity incrops is far less widespread than Zn deficiency. However, toxicitysymptoms usually become visible at Zn concentrations of more than 300 mgZn kg⁻¹ in the leaves. Some plants are known to be able to grow on soilthat has a high concentration of Zn, such as Silene vulgaris, Thlaspicaerulescens, Arabidopsis halleri and Viola calaminaria (see also Table3 in Broadley, M. R. et al., 2007, New Phytologist 173:677-702). It hasbeen suggested to use these Zn hyperaccumulators as sanitation plants toextract zinc from the soil, whereby the metal is concentrated in thebiomass, that can be harvested, incinerated and properly disposed of.Such a method, known as phytoremediation, is currently not attractive asthe known metal hyperaccumulator plants are relatively small, thusproducing not sufficient biomass to yield a high metal extractioncapacity.

Thus, there is still need to be able to control the adaptation of plantsto changes in zinc concentration in the environment.

SUMMARY OF THE INVENTION

The invention now relates to a method to change the capacity of a plantfor adaptation to changes in the zinc concentration in the environment,especially the soil, comprising providing said plant with a nucleotidecoding for a bZIP19 and/or bZIP23 protein or a functional equivalentthereof. Preferably in such a method the tolerance to Zn deficiency isincreased. In another embodiment in such a method said plant is capableof hyperaccumulating zinc. In a further embodiment said plant is capableof enhancing concentrations of zinc in edible parts.

In the above mentioned methods, the plant is preferably provided with anucleotide coding for a bZIP19 protein and a nucleotide coding for abZIP23 protein. More preferably, said plant is additionally providedwith one or more polynucleotides coding for a protein selected from thegroup consisting of heavy metal transporters, preferably HMA2, HMA3 orHMA4, YSL proteins, preferably YSL, preferably YSL1 or YSL3, ZIP or IRTproteins, ZIF proteins, NAS proteins, MRP proteins, FRD3 and MTPs.

The invention further relates to a plant transformed with apolynucleotide coding for a bZIP19 and/or a bZIP23 protein.

The invention further comprises a method according as described above ora plant as described above, wherein the polynucleotide is derived fromArabidopsis, more preferably A. thaliana.

The invention further relates to a plant made by a method according tothe invention, preferably wherein said plant overexpresses a bZIP19and/or a bZIP23 protein. Preferably said plant is tolerant to Zndeficiency. Alternatively, said plant is a Zn hyperaccumulator and/orhas an elevated amount of bioavailable Zn in its edible parts.

Also part of the invention is a method for phytoremediation, comprisinggrowing a plant according to the invention on soil that is polluted withZn, harvesting said plant and disposing of the biomass. Further part ofthe invention is a method for biofortification, comprising growing aplant according to to the invention on soil that is polluted with Zn,harvesting said plant and disposing of the biomass.

Also part of the invention is the use of the (nucleotides encoding) thebZIP19 and/or bZIP protein in the methods of the invention and/or forproducing the plants of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1: Amino acid sequences of Arabidopsis thaliana proteins bZIP 19(SEQ ID NO:1), bZIP23 (SEQ ID NO:2) and bZIP24 (SEQ ID NO:3). Aminoacids highlighted in green correspond to the bZIP domain. Amino acidshighlighted in grey correspond to two conserved motifs rich in histidineresidues. An asterisk or dot below the aligned sequences indicatesidentical, respectively, similar amino acids.

FIG. 2A-2B: Yeast-one-hybrid baits and F group bZIP gene expression.FIG. 2A Schematic diagram of the baits (A-G) used to construct thereporter vectors in the yeast-one-hybrid assay. The grey box representsthe 10-bp palindromic motif. FIG. 2B Relative transcript levels (RTL) ofbZIP19, bZIP23 and bZIP24 in 3-week-old Arabidopsis seedlings grown inMS medium, without (Zn−), with 30 μM (Zn+) or with 300 μM ZnSO₄ (Zn++).Error bars indicate SE.

FIG. 3A-3C: Gene expression and zinc transport of the Arabidopsis ZIP4zinc transporter. FIG. 3A Relative transcript levels (RTL) of ZIP4 in3-week-old Arabidopsis seedlings grown in MS medium, with 30 μM ZnSO₄(Zn+) or without (Zn−). Error bars indicate SE. FIG. 3B Growth ofzrt1zrt2 S. cerevisiae cells carrying either the empty vector (zrt1zrt2Ø) or expressing ZIP4 (zrt1zrt2 ZIP4) was assayed by spotting serialdilutions of cells (OD₆₀₀ is shown on the left) on SD-URA selectivemedium with 0.4 or 0.8 mM ZnCl₂. FIG. 3C OD measurements at theindicated time-intervals of zrt1zrt2 Ø and zrt1zrt2 ZIP4 on SD-URAselective liquid medium with 0.4 mM ZnCl₂. Error bars indicate SE.

FIG. 4A-4C: The double T-DNA insertion mutant m19m23 is hypersensitiveto zinc deficiency. FIG. 4A Effect of zinc deficiency on 3-week-oldseedlings of Arabidopsis (WT), bZIP19 T-DNA insertion mutants, m19,bZIP23 T-DNA insertion mutants, m23, and double T-DNA insertion mutants,m19m23, grown in MS medium without (Zn−) and with 30 μM ZnSO₄ (Zn+).FIG. 4B Effect of zinc supply on 4-week-old plants of Arabidopsis (WT),m19, m23 and m19m23, grown in hydroponics at 0.05 μM (Zn−), 2 μM (Zn+)and 25 μM ZnSO₄ (Zn++). FIG. 4C Zinc concentration, in mg kg⁻¹ dryweight, and dry weight (DW), in g, of roots (white bars) and shoots(grey bars) of 4-week-old WT, m19, m23 and m19m23 plants, grown inhydroponics at 0.05 μM ZnSO₄ (Zn−). *P<0.05, **P<0.01, ***P<0.001;representing significant differences of the mean in comparison with theWT mean. Error bars indicate SE.

FIG. 5A-5B: FIG. 5A Schematic drawing of the bZIP19 mutant allele (m19)and the bZIP23 mutant allele (m23). Triangles indicate T-DNA insertions.White boxes indicate exons, grey boxes indicate introns, lines indicate5′ and 3′ untranslated sequences. FIG. 5B Relative transcript levels(RTL) of bZIP19 (white bars) and bZIP23 (grey bars) in 3-week-oldseedlings of Arabidopsis wild type plants (WT), homozygous bZIP19 T-DNAinsertion mutants (m19), homozygous bZIP23 T-DNA insertion mutants(m23), and T-DNA insertion double mutants (m19m23) grown in MS medium.Error bars indicate SE.

FIG. 6: Zinc concentration (in mg kg⁻¹ dry weight) and dry weight (DW;in g) of roots (white bars) and shoots (grey bars) of 4-week-oldwild-type (WT), bZIP19 (m19) and bZIP23 (m23) single mutants and doublemutants (m19m23), grown in hydroponics at 2 μM ZnSO₄ (Zn+). Error barsindicate SE.

FIG. 7: Zinc concentration (in mg kg⁻¹ dry weight) and dry weight (DW;in g) of roots (white bars) and shoots (grey bars) of 4-week-oldwild-type (WT), bZIP19 (m19) and bZIP23 (m23) single mutants and doublemutants (m19m23), grown in hydroponics at 25 μM ZnSO₄ (Zn+). Error barsindicate SE.

FIG. 8A-8B: Complementation study and expression analysis of putativetarget genes. FIG. 8A Arabidopsis wild-type plants (WT), double T-DNAinsertion mutants (m19m23) and double mutants constitutively expressingeither bZIP19 (m19m23-OX19) or bZIP23 (m19m23-OX23), grown for 4 weeksin MS medium without (Zn−) and with 30 μM ZnSO₄ (Zn+). FIG. 8B Relativetranscript levels (RTL) of ZIP4, ZIP1, ZIPS, ZIPS, ZIP9, ZIP12, IRT3 andZIP2 in 3-week-old wild-type seedlings of Arabidopsis (WT) (white bars)and m19m23 double mutants (grey bars) grown in MS medium with 30 μMZnSO₄ (Zn+) or without (Zn−). Error bars indicate SE.

FIG. 9: Visible phenotypes of OX 19 (#19, 14, 15), OX 23 (#16, 17, 18)and untransformed Arabidopsis Col plants (WT), grown for 6 weeks onhydroponics medium to which no Zn has been added (0 μM Zn), creatingstrong zinc deficiency symptoms. Lines 19, 16 and 17 appear to havelarger rosettes and darker green leaves, showing less sensitivity tozinc deficiency, when compared to WT.

FIG. 10:: Total plant dry weight comparisons of OX 19 (#19, 14, 15), OX23 (#16, 17, 18) and untransformed Arabidopsis Col plants (WT), grownfor 6 weeks on hydroponics medium to which no Zn has been added (0 μMZn) (A, left panel); or on hydroponic medium containing normal Zn (2 μMZn) (B, right panel). Only for the low Zn treatment, line #16 was foundto be significantly different from WT.

FIG. 11: Zinc concentrations of OX 19 (#19, 14, 15), OX 23 (#16, 17, 18)and untransformed Arabidopsis Col plants (WT), grown for 6 weeks onhydroponics medium to which no Zn has been added (0 μM Zn).

FIG. 12: Visible phenotypes of pZIP4::bZIP19 (#4, 5, 6, 7, 8, 9) anduntransformed Arabidopsis Col plants (#1, 2, 3), grown for 3weeks onhydroponics medium to which no Zn has been added (0 μM Zn). Plants arenot yet showing Zn deficiency symptoms. Although there is some variationwithin lines due to plants that germinated later than the rest, ingeneral, pZIP4::bZIP19 plants are showing larger rosette diameters thanuntransformed plants.

FIG. 13A-D: DNA sequence and schematic drawing of the proZIP4::bZIP19construct (pBG0072) used to generate transgenic Arabidopsis expressingthe bZIP19 cDNA under control of the zinc deficiency responsive ZIP4promoter.

DEFINITIONS

The term “gene” is used broadly to refer to any segment of nucleic acidassociated with a biological function. Thus, genes include codingsequences and/or the regulatory sequences required for their expression.For example, gene refers to a nucleic acid fragment that expresses mRNAor functional RNA, or encodes a specific protein, and which includesregulatory sequences. Genes also include nonexpressed DNA segments that,for example, form recognition sequences for other proteins. Genes can beobtained from a variety of sources, including cloning from a source ofinterest or synthesizing from known or predicted sequence information,and may include sequences designed to have desired parameters.

The term “native” or “wild type” gene refers to a gene that is presentin the genome of an untransformed cell, i. e., a cell not having a knownmutation. The term “native” or “wild type” is intended to encompassallelic variants of the gene.

A “marker gene” encodes a selectable or screenable trait. The term“selectable marker” refers to a polynucleotide sequence encoding ametabolic trait which allows for the separation of transgenic andnon-transgenic organisms and mostly refers to the provision ofantibiotic resistance. A selectable marker is for example the aph (npt)encoded kanamycin resistance marker, or the hpt gene, the gene codingfor hygromycin resistance. Other selection markers are for instancereporter genes such as chloramphenicol acetyl transferase,β-galactosidase, luciferase and green fluorescence protein.Identification methods for the products of reporter genes include, butare not limited to, enzymatic assays and fluorimetric assays. Reportergenes and assays to detect their products are well known in the art andare described, for example in Current Protocols in Molecular Biology,eds. Ausubel et al., Greene Publishing and Wiley-Interscience: New York(1987) and periodic updates.

The term “chimeric gene” refers to any gene that contains 1) nucleotidesequences, including regulatory and coding sequences, that are not foundtogether in nature, or 2) nucleotide sequences encoding parts ofproteins not naturally adjoined, or 3) parts of promoters that are notnaturally adjoined. Accordingly, a chimeric gene may comprise regulatorysequences and coding sequences that are derived from different sources,or comprise regulatory sequences and coding sequences derived from thesame source, but arranged in a manner different from that found innature.

A “transgene” refers to a gene that has been introduced into the genomeby transformation and that preferably is stably maintained. Transgenesmay include, for example, genes that are either heterologous orhomologous to the genes of a particular plant to be transformed.Additionally, transgenes may comprise native genes inserted into anon-native organism or chimeric genes. The term “endogenous gene” refersto a native gene in its natural location in the genome of an organism. A“foreign” gene refers to a gene not normally found in the host organismbut that is introduced by gene transfer.

An “oligonucleotide”, e. g., for use in probing or amplificationreactions, may be about 30 or fewer nucleotides in length (e. g., 9, 12,15, 18, 20, 21 or 24, or any number between 9 and 30). Generallyspecific primers are upwards of 14 nucleotides in length. For optimumspecificity and cost effectiveness, primers of 16 to 24 nucleotides inlength may be preferred. Those skilled in the art are well versed in thedesign of primers for use in processes such as PCR. If required, probingcan be done with entire restriction fragments of the gene disclosedherein which may be 100's or even 1000's of nucleotides in length.

The terms “protein”, “peptide” and “polypeptide” are usedinterchangeably herein.

“Coding sequence” refers to a nucleotide (DNA or RNA) sequence thatcodes for a specific amino acid sequence and excludes the non-codingsequences. It may constitute an “uninterrupted coding sequence”, i. e.,lacking an intron, such as in a cDNA or it may include one or moreintrons bound by appropriate splice junctions. An “intron” is a sequenceof RNA which is contained in the primary transcript but which is removedthrough cleavage and re-ligation of the RNA within the cell to createthe mature mRNA that can be translated into a protein. Also the DNAcoding for said sequence of RNA is designated as “intron”. “Exons” arethe coding parts of the DNA or RNA sequence, which are separated fromeach other by introns.

“Regulatory sequences” refer to nucleotide sequences located upstream(5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influence the transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences include enhancers, promoters, translationleader sequences, introns, and polyadenylation signal sequences. Theyinclude natural and synthetic sequences as well as sequences which maybe a combination of synthetic and natural sequences. As is noted above,the term “suitable regulatory sequences” is not limited to promoters.

“Promoter” refers to a nucleotide sequence, usually upstream (5′) to itscoding sequence, which controls the expression of the coding sequence byproviding the recognition for RNA polymerase and other factors requiredfor proper transcription. “Promoter” includes a minimal promoter that isa short DNA sequence comprised of a TATA box and other sequences thatserve to specify the site of transcription initiation, to whichregulatory elements are added for control of expression. “Promoter” alsorefers to a nucleotide sequence that includes a minimal promoter plusregulatory elements that is capable of controlling the expression of acoding sequence or functional RNA. This type of promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is aDNA sequence which can stimulate promoter activity and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue specificity of a promoter. It is capable ofoperating in both orientations (normal or flipped), and is capable offunctioning even when moved either upstream or downstream from thepromoter. Both enhancers and other upstream promoter elements bindsequence-specific DNA-binding proteins that mediate their effects.Promoters may be derived in their entirety from a native gene, or becomposed of different elements derived from different promoters found innature, or even be comprised of synthetic DNA segments. A promoter mayalso contain DNA sequences that are involved in the binding of proteinfactors which control the effectiveness of transcription initiation inresponse to physiological or developmental conditions.

The “initiation site” is the position surrounding the first nucleotidethat is part of the transcribed sequence, which is also defined asposition +1. With respect to this site all other sequences of the geneand its controlling regions are numbered. Downstream sequences (i. e.,further protein encoding sequences in the 3′ direction) are denominatedpositive, while upstream sequences (mostly of the controlling regions inthe 5′direction) are denominated negative.

“Constitutive expression” refers to expression using a constitutivepromoter.

“Conditional” and “regulated expression” refer to expression controlledby a regulated promoter.

“Constitutive promoter” refers to a promoter that is able to express theopen reading frame (ORF) that it controls in all or nearly all of theplant tissues during all or nearly all developmental stages of theplant.

The terms “open reading frame” and “ORF” refer to the amino acidsequence encoded between translation initiation and termination codonsof a coding sequence. The terms “initiation codon” and “terminationcodon” refer to a unit of three adjacent nucleotides ('codon') in acoding sequence that specifies initiation and chain termination,respectively, of protein synthesis (mRNA translation).

“Regulated promoter” refers to promoters that direct gene expression notconstitutively, but in a temporally-and/or spatially-regulated manner,and includes both tissue-specific and inducible promoters. It includesnatural and synthetic sequences as well as sequences which may be acombination of synthetic and natural sequences. Different promoters maydirect the expression of a gene in different tissues or cell types, orat different stages of development, or in response to differentenvironmental conditions. New promoters of various types useful in plantcells are constantly being discovered, numerous examples may be found inthe compilation by Okamuro, J. K. and Goldberg, R. B. (1989) Chapter 1,“Regulation of Plant Gene Expression: General Principles” in Stumpf, P.K. and Conn, E. E. Eds., The Biochemistry of Plants: A comprehensivetreatise. Academic Press, N.Y. USA).

“Tissue-specific promoter” refers to regulated promoters that are notexpressed in all plant cells but only in one or more cell types inspecific organs (such as roots, stems, leaves or seeds), specifictissues (such as embryo or cotyledon), or specific cell types (such asleaf parenchyma or seed storage cells).

“Operably-linked” refers to the association of nucleic acid sequences ona single nucleic acid fragment so that the function of one is affectedby the other. For example, a regulatory DNA sequence is said to be“operably linked to” or “associated with” a DNA sequence that codes foran RNA or a polypeptide if the two sequences are situated such that theregulatory DNA sequence affects expression of the coding DNA sequence(i. e., that the coding sequence or functional RNA is under thetranscriptional control of the promoter). Coding sequences can beoperably-linked to regulatory sequences in sense or antisenseorientation.

“Expression” refers to the transcription and/or translation of anendogenous gene, ORF or portion thereof, or a transgene in plants.Expression refers to the transcription and stable accumulation of sense(mRNA) or functional RNA. Expression may also refer to the production ofprotein.

“Specific expression” is the expression of gene products which islimited to one or a few plant tissues (spatial limitation) and/or to oneor a few plant developmental stages (temporal limitation). It isacknowledged that hardly a true specificity exists: promoters seem topreferably switch on in some tissues, while in other tissues there canbe no or only little activity. This phenomenon is known as leakyexpression. However, with tissue-specific expression in this inventionis meant preferable expression in one or a few plant tissues.

The terms “heterologous DNA sequence”, “exogenous DNA segment” or“heterologous nucleic acid”, as used herein, each refer to a sequencethat originates from a source foreign to the particular host cell or, iffrom the same source, is modified from its original form. Thus, aheterologous gene in a host cell includes a gene that is endogenous tothe particular host cell but has been modified through, for example, theuse of DNA shuffling. The terms also include non-naturally occurringmultiple copies of a naturally occurring DNA sequence. Thus, the termsrefer to a DNA segment that is foreign or heterologous to the cell, orhomologous to the cell but in a position within the host cell nucleicacid in which the element is not ordinarily found. Exogenous DNAsegments are expressed to yield exogenous polypeptides. A “homologous”DNA sequence is a DNA sequence that is naturally associated with a hostcell into which it is introduced.

“Homologous to” in the context of nucleotide or amino acid sequenceidentity refers to the similarity between the nucleotide sequence of twonucleic acid molecules or between the amino acid sequences of twoprotein molecules. Estimates of such homology are provided by eitherDNA-DNA or DNA-RNA hybridization under conditions of stringency as iswell understood by those skilled in the art (as described in Haines andHiggins (eds.), Nucleic Acid Hybridization, IRL Press, Oxford, U. K.),or by the comparison of sequence similarity between two nucleic acids orproteins. Two nucleotide or amino acid sequences are homologous whentheir sequences have a sequence similarity of more than 60%, preferablymore than 70%, 80%, 85%, 90%, 95%, or even 98%.

The term “substantially similar” refers to nucleotide and amino acidsequences that represent functional and/or structural equivalents ofsequences disclosed herein. For example, altered nucleotide sequenceswhich simply reflect the degeneracy of the genetic code but nonethelessencode amino acid sequences that are identical to a particular aminoacid sequence are substantially similar to the particular sequences. Inaddition, amino acid sequences that are substantially similar to aparticular sequence are those wherein overall amino acid identity is atleast 65% or greater to the instant sequences. Modifications that resultin equivalent nucleotide or amino acid sequences are well within theroutine skill in the art. Moreover, the skilled artisan recognizes thatequivalent nucleotide sequences encompassed by this invention can alsobe defined by their ability to hybridize, under low, moderate and/orstringent conditions (e. g., 0. 1× SSC, 0.1% SDS, 65° C.), with thenucleotide sequences that are within the literal scope of the instantclaims.

The term “transformation” refers to the transfer of a nucleic acidfragment into the genome of a host cell, resulting in genetically stableinheritance. Host cells containing the transformed nucleic acidfragments are referred to as “transgenic” cells, and organismscomprising transgenic cells are referred to as “transgenic organisms”.Examples of methods of transformation of plants and plant cells includeAgrobacterium-mediated transformation (De Blaere et al., 1987) particlebombardment technology (Klein et al. 1987; U.S. Pat. No. 4,945,050),microinjection, CaPO₄ precipitation, lipofection (liposome fusion), useof a gene gun and DNA vector transporter (Wu et al., 1992). Whole plantsmay be regenerated from transgenic cells by methods well known to theskilled artisan (see, for example, Fromm et al., 1990).

“Transformed”, “transgenic” and “recombinant” refer to a host organismsuch as a bacterium or a plant into which a heterologous nucleic acidmolecule has been introduced. The heterologous nucleic acid molecule canbe stably integrated into the genome generally known in the art and aredisclosed in Sambrook et al., 1989. See also Innis et al., 1995 andGelfand, 1995; and Innis and Gelfand, 1999. For example, “transformed”,“transformant”, and “transgenic” plants or calli have been through thetransformation process and contain a foreign gene integrated into theirchromosome. The term “untransformed” refers to normal plants that havenot been through the transformation process.

“Transiently transformed” refers to cells in which transgenes andforeign DNA have been introduced (for example, by such methods asAgrobacterium-mediated transformation or biolistic bombardment), but notselected for stable maintenance.

“Stably transformed” refers to cells that have been selected andregenerated on a selection media following transformation.

“Genetically stable” and “heritable” refer to chromosomally-integratedgenetic elements that are stably maintained in the plant and stablyinherited by progeny through successive generations.

“Chromosomally-integrated” refers to the integration of a foreign geneor DNA construct into the host DNA by covalent bonds. Where genes arenot “chromosomally integrated” they may be “transiently expressed”.Transient expression of a gene refers to the expression of a gene thatis not integrated into the host chromosome but functions independently,either as part of an autonomously replicating plasmid or expressioncassette, for example, or as part of another biological system such as avirus.

“Primary transformant” refers to transgenic plants that are of the samegenetic generation as the tissue which was initially transformed (i. e.,not having gone through meiosis and fertilization since transformation).

“Secondary transformants” and the “T1, T2, T3, etc. generations” referto transgenic plants derived from primary transformants through one ormore meiotic and fertilization cycles. They may be derived byself-fertilization of primary or secondary transformants or crosses ofprimary or secondary transformants with other transformed oruntransformed plants. Secondary transformants are an aspect of thepresent invention.

“Genome” refers to the complete genetic material of an organism.

The term “nucleic acid” or “polynucleotide” refers todeoxyribonucleotides or ribonucleotides and polymers thereof in eithersingle-or double-stranded form, composed of monomers (nucleotides)containing a sugar, phosphate and a base which is either a purine orpyrimidine. Unless specifically limited, the term encompasses nucleicacids containing known analogs of natural nucleotides which have similarbinding properties as the reference nucleic acid and are metabolized ina manner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (e. g., degeneratecodon substitutions) and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., 1991; Ohtsuka et al., 1985;Rossolini et al. 1994). A “nucleic acid fragment” is a fraction of agiven nucleic acid molecule. In higher plants, deoxyribonucleic acid(DNA) is the genetic material while ribonucleic acid (RNA) is involvedin the transfer of information contained within DNA into proteins. Theterm “nucleotide sequence” refers to a polymer of DNA or RNA which canbe single-or double-stranded, optionally containing synthetic,non-natural or altered nucleotide bases capable of incorporation intoDNA or RNA polymers. The terms “nucleic acid” or “nucleic acid sequence”may also be used interchangeably with gene, cDNA, DNA and RNA encoded bya gene.

The nucleotide sequences used in aspects of the invention include boththe naturally occurring sequences as well as mutant (variant) forms.Such variants will continue to possess the desired activity, i. e.,either promoter activity or the activity of the product encoded by theopen reading frame of the non-variant nucleotide sequence.

Thus, by “variant” is intended a substantially similar sequence. Fornucleotide sequences comprising an open reading frame, variants includethose sequences that, because of the degeneracy of the genetic code,encode the identical amino acid sequence of the native protein.Naturally occurring allelic variants such as these can be identifiedwith the use of well-known molecular biology techniques, as, forexample, with polymerase chain reaction (PCR) and hybridizationtechniques. Variant nucleotide sequences also include syntheticallyderived nucleotide sequences, such as those generated, for example, byusing site-directed mutagenesis and for open reading frames, encode thenative protein, as well as those that encode a polypeptide having aminoacid substitutions relative to the native protein. Generally, nucleotidesequence variants of the invention will have at least 40, 50, 60, to70%, e. g., preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%,generally at least 80%, e. g., 81%-84%, at least 85%, e. g., 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98% and 99%nucleotide sequence identity to the native (wild type or endogenous)nucleotide sequence.

The term “nucleotide sequence identity” or “nucleotide sequencehomology” as used herein denotes the level of similarity, respectivelythe level of homology, between two polynucleotides. Polynucleotides have“identical” sequences if the sequence of nucleotides in the twosequences is the same. Polynucleotides have “homologous” sequences ifthe sequence of nucleotides in the two sequences is the same whenaligned for maximum correspondence. Sequence comparison between two ormore polynucleotides is generally performed by comparing portions of thetwo sequences over a comparison window to identify and compare localregions of sequence similarity. The comparison window is generally fromabout 20 to 200 contiguous nucleotides. The “percentage of sequenceidentity ” or “percentage of sequence homology” for polynucleotides,such as 50, 60, 70, 80, 90, 95, 98, 99 or 100 percent sequence identityor homology may be determined by comparing two optimally alignedsequences over a comparison window, wherein the portion of thepolynucleotide sequence in the comparison window may include additionsor deletions (i.e. gaps) as compared to the reference sequence (whichdoes not comprise additions or deletions) for optimal alignment of thetwo sequences. The percentage is calculated by: (a) determining thenumber of positions at which the identical nucleic acid base occurs inboth sequences to yield the number of matched positions; (b) dividingthe number of matched positions by the total number of positions in thewindow of comparison; and (c) multiplying the result by 100 to yield thepercentage of sequence homology. Optimal alignment of sequences forcomparison may be conducted by computerized implementations of knownalgorithms, or by visual inspection. Readily available sequencecomparison and multiple sequence alignment algorithms are, respectively,the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990;Altschul et al., 1997) and ClustalW programs, both available on theinternet. Other suitable programs include, but are not limited to, GAP,BestFit, PlotSimilarity, and FASTA in the Wisconsin Genetics SoftwarePackage (Genetics Computer Group (GCG), Madison, Wis., USA) (Devereux etal., 1984).

The nucleic acid sequences of the invention can be “optimized” forenhanced expression in plants of interest. See, for example, EP 0359472or WO 91/16432. In this manner, the open reading frames in genes or genefragments can be synthesized utilizing plant-preferred codons. Thus, thenucleotide sequences can be optimized for expression in any plant.

By “variant” polypeptide is intended a polypeptide derived from thenative protein by deletion (so-called truncation) or addition of one ormore amino acids to the N-terminal and/or C-terminal end of the nativeprotein; deletion or addition of one or more amino acids at one or moresites in the native protein; or substitution of one or more amino acidsat one or more sites in the native protein. Such variants may resultfrom, for example, genetic polymorphism or from human manipulation.Methods for such manipulations are generally known in the art. Alsoincluded in the definition of variant polypeptide are “orthologous”polypeptides (orthologs), which are peptides encoded by genes indifferent species that evolved from a common ancestral gene byspeciation. Normally, orthologs retain the same function in the courseof evolution

Thus, the polypeptides may be altered in various ways including aminoacid substitutions, deletions, truncations, and insertions. Methods forsuch manipulations are generally known in the art. Conservativesubstitutions, such as exchanging one amino acid with another havingsimilar properties, are preferred. Individual substitutions deletions oradditions that alter, add or delete a single amino acid or a smallpercentage of amino acids (typically less than 5%, more typically lessthan 1%) in an encoded sequence are “conservatively modifiedvariations”, where the alterations result in the substitution of anamino acid with a chemically similar amino acid.

“Expression cassette” as used herein means a DNA sequence capable ofdirecting expression of a particular nucleotide sequence in anappropriate host cell, comprising a promoter operably linked to thenucleotide sequence of interest which is operably linked to terminationsignals. It also typically comprises sequences required for propertranslation of the nucleotide sequence. The coding region usually codesfor a protein of interest but may also code for a functional RNA ofinterest, for example antisense RNA or a nontranslated RNA, in the senseor antisense direction. The expression cassette comprising thenucleotide sequence of interest may be chimeric, meaning that at leastone of its components is heterologous with respect to at least one ofits other components. The expression cassette may also be one which isnaturally occurring but has been obtained in a recombinant form usefulfor heterologous expression. The expression of the nucleotide sequencein the expression cassette may be under the control of a constitutivepromoter or of an inducible promoter which initiates transcription onlywhen the host cell is exposed to some particular external stimulus. Inthe case of a multicellular organism, the promoter can also be specificto a particular tissue or organ or stage of development.

The term “vector” as used herein refers to a construction comprised ofgenetic material designed to direct transformation of a targeted cell. Avector contains multiple genetic elements positionally and sequentiallyoriented, i.e., operatively linked with other necessary elements suchthat the nucleic acid in a nucleic acid cassette can be transcribed andwhen necessary, translated in the transformed cells. “Vector” is definedto include, inter alia, any plasmid, cosmid, phage or Agrobacteriumbinary vector in double or single stranded linear or circular form whichmay or may not be self transmissible or mobilizable, and which cantransform prokaryotic or eukaryotic host either by integration into thecellular genome or exist extrachromosomally (e. g. autonomousreplicating plasmid with an origin of replication). Specificallyincluded are shuttle vectors by which is meant a DNA vehicle capable,naturally or by design, of replication in two different host organisms,which may be selected from actinomycetes and related species, bacteriaand eukaryotic (e. g. higher plant, mammalian, yeast or fungal cells).Preferably the nucleic acid in the vector is under the control of, andoperably linked to, an appropriate promoter or other regulatory elementsfor transcription in a host cell such as a microbial, e. g. bacterial,or plant cell. The vector may be a bi-functional expression vector whichfunctions in multiple hosts. In the case of genomic DNA, this maycontain its own promoter or other regulatory elements and in the case ofcDNA this may be under the control of an appropriate promoter or otherregulatory elements for expression in the host cell.

“Cloning vectors” typically contain one or a small number of restrictionendonuclease recognition sites at which foreign DNA sequences can beinserted in a determinable fashion without loss of essential biologicalfunction of the vector, as well as a marker gene that is suitable foruse in the identification and selection of cells transformed with thecloning vector.

A “transgenic plant” is a plant having one or more plant cells thatcontain an expression vector.

“Plant tissue” includes differentiated and undifferentiated tissues orplants, including but not limited to roots, stems, shoots, leaves,pollen, seeds, tumor tissue and various forms of cells and culture suchas single cells, protoplast, embryos, and callus tissue. The planttissue may be in plants or in organ, tissue or cell culture.

The term “plant,” as used herein, refers to any type of plant. Theinventors have provided below an exemplary description of some plantsthat may be used with the invention. However, the list is provided forillustrative purposes only and is not limiting, as other types of plantswill be known to those of skill in the art and could be used with theinvention.

A common class of plants exploited in agriculture are vegetable crops,including artichokes, kohlrabi, arugula, leeks, asparagus, lettuce(e.g., head, leaf, romaine), bok choy, malanga, broccoli, melons (e.g.,muskmelon, watermelon, crenshaw, honeydew, cantaloupe), brusselssprouts, cabbage, cardoni, carrots, napa, cauliflower, okra, onions,celery, parsley, chick peas, parsnips, chicory, Chinese cabbage,peppers, collards, potatoes, cucumber plants (marrows, cucumbers),pumpkins, cucurbits, radishes, dry bulb onions, rutabaga, eggplant,salsify, escarole, shallots, endive, garlic, spinach, green onions,squash, greens, beet (sugar beet and fodder beet), sweet potatoes,swiss-chard, horseradish, tomatoes, kale, turnips, and spices.

Other types of plants frequently finding commercial use include fruitand vine crops such as apples, apricots, cherries, nectarines, peaches,pears, plums, prunes, quince almonds, chestnuts, filberts, pecans,pistachios, walnuts, citrus, blueberries, boysenberries, cranberries,currants, loganberries, raspberries, strawberries, blackberries, grapes,avocados, bananas, kiwi, persimmons, pomegranate, pineapple, tropicalfruits, pornes, melon, mango, papaya, and lychee.

Many of the most widely grown plants are field crop plants such asevening primrose, meadow foam, corn (field, sweet, popcorn), hops,jojoba, peanuts, rice, safflower, small grains (barley, oats, rye,wheat, etc.), sorghum, tobacco, kapok, leguminous plants (beans,lentils, peas, soybeans), oil plants (rape, mustard, poppy, olives,sunflowers, coconut, castor oil plants, cocoa beans, groundnuts), fiberplants (cotton, flax, hemp, jute), Lauraceae (cinnamon, camphor), orplants such as coffee, sugarcane, tea, and natural rubber plants.

Especially applicable in the present invention are plants with a highbiomass, such as plants that are also used in biofuel production, suchas Miscanthus, switchgrass, poplar, eucalyptus, loblolly pine, willow,silver maple, alfalfa, Jatropha, and Pongamia pinnata.

Another economically important group of plants are ornamental plants.Examples of commonly grown ornamental plants include Alstroemeria (e.g.,Alstoemeria brasiliensis), aster, azalea (e.g., Rhododendron sp.),begonias (e.g., Begonia sp.), bellflower, bouganvillea, cactus (e.g.,Cactaceae schlumbergera truncata), camellia, carnation (e.g., Dianthuscaryophyllus), chrysanthemums (e.g., Chrysanthemum sp.), clematis (e.g.,Clematis sp.), cockscomb, columbine, cyclamen (e.g., Cyclamen sp.),daffodils (e.g., Narcissus sp.), false cypress, freesia (e.g., Freesiarefracta), geraniums, gerberas, gladiolus (e.g., Gladiolus sp.), holly,hibiscus (e.g., Hibiscus rosasanensis), hydrangea (e.g., Macrophyllahydrangea), juniper, lilies (e.g., Lilium sp.), magnolia, miniroses,orchids (e.g., members of the family Orchidaceae), petunias (e.g.,Petunia hybrida), poinsettia (e.g., Euphorbia pulcherima), primroses,rhododendron, roses (e.g., Rosa sp.), snapdragons (e.g., Antirrhinumsp.), shrubs, trees such as forest (broad-leaved trees and evergreens,such as conifers) and tulips (e.g., Tulipa sp.).

The term “plant part”, as used herein, includes reference to, but is notlimited to, single cells and tissues from microspores, pollen, ovules,leaves, embryos, roots, root tips, anthers, flowers, fruits, seeds,stems, shoots, scions, rootstocks, protoplasts, calli, meristematictissues and the like.

The term “crop plant”, as used herein, refers to a plant which isharvested or provides a harvestable product.

The terms “seedling” and “plantlet”, as used herein, are interchangeableand refer to the juvenile plant grown from a sprout, embryo or agerminating seed and generally include any small plants showing welldeveloped green cotyledons and root elongation and which are propagatedprior to transplanting in the ultimate location wherein they are tomature.

The term “tissue culture”, as used herein, refers to a culture of plantcells wherein the cells are propagated in a nutrient medium undercontrolled conditions.

“Significant increase” is an increase that is larger than the margin oferror inherent in the measurement technique, preferably an increase byabout 10%-50%, or even 2-fold or greater.

“Significantly less” means that the decrease is larger than the marginof error inherent in the measurement technique, preferably a decrease byabout 2-fold or greater.

The term “endogenous” as in “endogenously produced” refers to producedwithin the plant (cell).

The term “production area”, as used herein, refers to a location whereplants are grown and where products in the form of plants or plant partsare produced for harvest. The size of the production area is generallyexpressed in square meters or acres of land. A production area can be anopen field or a greenhouse.

The term “biomass production”, as used herein, refers to the productionof plant derived organic material.

The term “dry matter content”, as used herein, refers to the massfraction (%) that remains after the water fraction (%) has been removedby drying.

DNA Sequences for Transformation

Virtually any DNA composition may be used for delivery to recipientplant cells, to ultimately produce fertile transgenic plants inaccordance with the present invention. For example, DNA segments in theform of vectors and plasmids, or linear DNA fragments, in some instancescontaining only the DNA element to be expressed in the plant, and thelike, may be employed. The construction of vectors which may be employedin conjunction with the present invention will be known to those ofskill of the art in light of the present disclosure (see, e. g.,Sambrook et al., 1989; Gelvin et al., 1990). Vectors, includingplasmids, cosmids, YACs (yeast artificial chromosomes), BACs (bacterialartificial chromosomes) and DNA segments for use in transforming cells,according to the present invention will, of course, comprise the cDNA,gene or genes necessary for production of isoprene in the transformant.

The vector of the invention can be introduced into any plant. The genesand sequences to be introduced can be conveniently used in expressioncassettes for introduction and expression in any plant of interest. Suchexpression cassettes will comprise a transcriptional initiation region(a promoter) linked to the gene encoding the isoprene synthase gene ofinterest. Such an expression cassette is preferably provided with aplurality of restriction sites for insertion of the gene of interest tobe under the transcriptional regulation of the regulatory regions, suchas the designated promoter. The expression cassette may additionallycontain selectable marker genes suitable for the particular hostorganism.

The transcriptional cassette will include in the 5′-to-3′ direction oftranscription, transcriptional and translational initiation regions, aDNA sequence of interest, and transcriptional and translationaltermination regions functional in plants.

The termination region may be native with the transcriptional initiationregion, may be native with the DNA sequence of interest, or may bederived from another source.

Convenient termination regions are available from the Ti-plasmid of A.tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also, Guerineau et al. (1991) Mol. Gen. Genet.262: 141-144; Proudfoot (1991) Cell 64: 671-674; Sanfacon et al. (1991)Genes Dev. 5: 141-149; Mogen et al. (1990) Plant Cell 2: 1261-1272;Munroe et al. (1990) Gene 91: 151-158; Ballas et al. (1989) NucleicAcids Res. 17: 7891-7903; Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

Methodologies for the construction of plant transformation constructsare described in the art.

In one embodiment of the invention, plants are transformed with thenucleotide coding for bZIP19 or bZIP23. These genes can be derived fromArabidopsis. Alternatively, orthologs of bZIP19 and bZIP23 may be used,such as those from Populus trichocarpa (PtrbZIP38 or XM_002305485.1 andPtrbZIP39 or XM_002313671.1); rice (OsbZIP48 or AK071639.1), Helianthusannuus (CD852649.1), Medicago truncatula (TA25329_3880). Solanumtuberosum (TA33339_3880), Glycine max (TA50226_3847; TA50224_3847),Sorghum bicolor (TA23717_4558), Zea mays (TA111061_4577; TA111059_4577;C0451643), or Hordeum vulgare (TA45897_4513).

The amino acid sequences of the Arabidopsis proteins are provided inFIG. 1, the genes for these proteins can be found in the gene databasesunder Accession No's At4g35040 or NM_119670.4 (bZIP19) and At2g16770 orNM_119670.4 (bZIP23). The sequences are also listed as SEQ ID NO: 2 andSEQ ID NO: 1, respectively. All bZIP proteins contain a characteristicand highly conserved basic domain, which binds DNA, and a leucine zipperdimerization motif. bZIPs can form homo and/or heterodimers, which bindDNA in a sequence-specific manner and are capable of binding shortpalindromic or pseudo-palindromic target sequences (Fyjii., Y. et al.,2000, Nature 7:889-893). Plant bZ1Ps are important for the regulation ofpathogen defence, environmental signalling and development, but so farno function is assigned to about two thirds of the bZIP members (Hakoby,M. et al., 2002, Trends Plant Sci. 7:106-111). Among these are thebZIP19 and bZIP23 genes. They belong to group F, one of ten groups inwhich this family is divided. This group contains a third member,bZIP24, not identified in the yeast-one-hybrid assay. bZIP19 and bZIP23predicted protein sequences share 69% of amino acid sequence identityand only 28 and 32%, respectively, with bZIP24. All three members of theF group contain two characteristic histidine-rich motifs (FIG. 1).

As can be seen, bZIP19 and bZIP23 share the bZIP domain at amino acid79-110 (bZIP23) and amino acid 94-125 (bZIP19) and two histidine-richmotifs at amino acid 36-48 and 51-60 (bZIP23) and amino acid 44-56 and59-68 (bZIP19).

Although it is possible to obtain overexpression of either bZIP19 orbZIP23 in a plant by providing such a plant with the above mentionednucleotide sequences, a preferred embodiment of the invention is a plantin which both proteins are overexpressed. As is exemplified in theexperimental part, the genes act redundantly, with bZIP19 only beingpartially redundant. bZIP transcription factors generally act as dimers.Their redundancy suggests that bZIP19 and bZIP23 act as homo(di)mers, inline with previous predictions (Deppmann, C. D. et al., 2006, Mol. Biol.Evol. 23:1480-1492).

Overexpression of bZIP19 and/or bZIP23 can be achieved by insertion ofone or more than one extra copy of the selected gene. It is not unknownfor plants or their progeny, originally transformed with one or morethan one extra copy of a nucleotide sequence to exhibit overexpression.

Obtaining sufficient levels of transgene expression in the appropriateplant tissues is an important aspect in the production of geneticallyengineered crops. Expression of heterologous DNA sequences in a planthost is dependent upon the presence of an operably linked promoter thatis functional within the plant host. Choice of the promoter sequencewill determine when and where within the organism the heterologous DNAsequence is expressed. The proteins of the current invention arepreferably expressed in the roots, where Zn is taken up from the soiland transported, and in other cells of the plant that are currentlyinvolved in transport and accumulation of zinc. Such root-specificpromoters are well known to a person skilled in the art and can bechosen from the the RolD, RPL16A. Tub-1, ARSK1, PsMT_(a) (WO97/20057),and Ataol promoter (Moller, S. G. and McPherson, M. J., 1998, The PlantJ., 13:781-791), the AAP6 promoter (Okumoto, S. et al., 2002, J. Biol.Chem. 277:54338-54346), the tobacco RB7 promoter (Yamamoto, Y. T. etal., 1991, Plant Cell 3:371-382.), the Arabidopsis ADH promoter(McKendree, W .L. et al., 1992, Plant Mol Biol. 19:859-862), theArabidopsis PHT1 promoter (Koyama, T. et al., 2004, J. Biosci. Bioeng.99:38-42), the Arabidopsis pyk10 promoter (Nitz, I. et al., 2001, PlantSci. 161:337-346), the peroxidase gene promoter (prxEa) of Arabidopsis(Wanapu and Shinmyo, 1996, Ann N. Y. Acad. Sci. 782: 107-114). thetomato SIREO promoter (Jones, M. O. et al., 2008, Funct. Plant Biol.35:1224-1233), the alfalfa MsPRP2 promoter (Winicov, I. et al.), therice ZRP3 and ZRP4 promoters (ref?), the maize RCc2 and RCc3 promoters(ref?), the strawberry FaRB7 promoter (Vaughan, S. P. et al., 2006, J.Exp. Botany doi: 10.1093/jxb/er1185) the promoters as disclosed in U.S.Pat. Nos. 5,459,252, 5,837,876, WO 97/005261, WO 2001/53502,WO2001/044454, WO 2006/024291 and WO 2006/022467. Alternatively,promoters can be used from genes that are normally controlled inexpression by bZIP19 or bZIP23, for example those as listed in Table 3.It is believed that these promoters are especially preferable since theyare expressed in the cells where the proteins normally act and sincethey will be induced by the presence of Zn and thus they will amplifythe Zn deficiency signal causing an earlier and particularly strongerresponse.

Although it is known that some transgenic approaches do indeed influenceadaptation of plants to changes in the zinc concentrations in theenvironment, these approaches are all attempts to makes plantstransgenic for zinc transporter proteins (such as ZAT, see van der Zaal,B. J., 1999, Plant Physiol. 119:1047-1055; AtHMA4, see Verret, F. etal., 2004, FEBS Lett 576:306-312; and NAS, see Takahashi, M. et al.,2003, Plant Cell 15:1263-1280). The current invention attempts tointerfere with the regulation of zinc, uptake, transport and storage inthe plant, by modulating the expression of genes that are regulators of,amongst others, these transport proteins. Thus, the invention interferesat a more basal level, which has the advantage that it is lessinfluenced by feedback mechanisms and other regulatory processesinvolved in zinc processing.

Although it is demonstrated in the experimental part that bZIP19 andbZIP23 play a major role in the control of adaptation to changes in thezinc content of the soil and although transforming plants with either orboth of those proteins would make the plants able to adapt to Zndeficiency or to act as Zn (hyper)accumulators, these properties can beenhanced by overexpressing additional proteins that are known to beinvolved in Zn homeostasis. These proteins are preferably chosen fromheavy metal transporters, preferably HMA2, HMA3 or HMA4, YSL proteins,preferably YSL, preferably YSL1 or YSL3, ZIP or IRT proteins, ZIFproteins, NAS proteins, MRP proteins, FRD3 and MTPs (metal transportingproteins).

As can be seen in this Table, genes encoding these proteins areavailable to the person skilled in the art and transformation of thesequences coding for these genes can be performed according to themethods as described herein.

TABLE A Proteins involved in Zn uptake, transport and storage NameDescription Acc. No. Gene Acc. No. Protein HMA2 cadmium-transportingATPase NM_119157.3 NP_194740.1 HMA3 potential Zn/Cd heavy metalAY434729.1 AAR10768.1 transporter HMA4 cadmium ion transmembraneNM_127468.4 NP_179501.1 transporter/cadmium- transporting ATPase/zincion transmembrane transporter YSL1 Oligopeptide transporter NM_118544.3NP_567694.2 YSL2 Oligopeptide transporter NM_122346.3 NP_197826.2 YSL3Oligopeptide transporter NM_124735.2 NP_200167.2 ZIP1 zinc iontransmembrane NM_112111.3 NP_187881.1 transporter ZIP2 zinc iontransmembrane NM_125344.2 NP_200760.1 transporter ZIP3 zinc iontransmembrane NM_128786.3 NP_180786.1 transporter ZIP4 copper iontransmembrane NM_100972.4 NP_172566.2 transporter ZIP5 metal iontransmembrane NM_202033.1 NP_973762.1 transporter ZIP6 metal iontransmembrane NM_128563.1 NP_180569.1 transporter ZIP7 metal iontransmembrane NM_126440.2 NP_178488.1 transporter ZIP8 zinc iontransmembrane NM_001161290.1 NP_001154762.1 transporter ZIP9 zinc iontransmembrane NM_119456.1 NP_195028.1 transporter ZIP10 zinc iontransmembrane NM_102864.2 NP_174411.2 transporter ZIP11 metal iontransmembrane NM_104468.2 NP_564703.1 transporter ZIP12 zinc iontransmembrane NM_125609.1 NP_201022.1 transporter IRT3 metal iontransmembrane NM_104776.4 NP_564766.1 transporter ZIF1 carbohydratetransmembrane NM_121377.4 NP_196878.2 transporter NAS1 nicotianaminesynthase NM_120577.3 NP_196114.1 NAS2 nicotianamine synthase NM_124990.1NP_200419.1 NAS3 nicotianamine synthase NM_100794.3 NP_172395.1 NAS4nicotianamine synthase NM_104521.2 NP_176038.1 MRP3 glutathioneS-conjugate- NM_112147.2 NP_187915.1 exporting ATPase FRD3antiporter/transporter NM_111683.1 NP_187461.1 MTP1 zinc iontransmembrane NM_180128.2 NP_850459.1 transporter MTP2 NM_116059.2NP_191753.1 MTP3 zinc ion transmembrane NM_115743.3 NP_191440.2transporter MTP8 metal tolerance protein NM_115668.2 NP_191365.2

(Over)expression of bZIP19 and/or bZ1P23 in plants together with one ormore of the above mentioned proteins would yield plants which would beable to uptake more zinc than a normal wild-type plant or to respondfaster to local deficiencies in zinc supply. By these characteristics,such plants would be very suitable to grow on high zinc containing soiland would therefore be able to act as phytoremediators, clearing thesoil from the toxic metal, or they would be able to grow on normal orlow zinc containing soil, but still have dietarily sufficient amounts ofzinc in their edible parts, or be less sensitive to zinc deficiencies.

Vectors for use in tissue-specific targeting of genes in transgenicplants will typically include tissue-specific promoters and may alsoinclude other tissue-specific control elements such as enhancersequences.

Additionally, vectors may be constructed and employed in theintracellular targeting of a specific gene product within the cells of atransgenic plant. This will generally be achieved by joining a DNAsequence encoding a transit or signal peptide sequence to the codingsequence of a particular gene. The resultant transit, or signal, peptidewill transport the protein to a particular intracellular, orextracellular destination, respectively, and will then bepost-translationally removed. Transit or signal peptides act byfacilitating the transport of proteins through intracellular membranes,e. g., vacuole, vesicle, plastid and mitochondrial membranes, whereassignal peptides direct proteins through the extracellular membrane.

A particular example of such a use concerns the direction of a protein(enzyme) to a particular organelle such as the vacuole rather than tothe cytoplasm. Signal peptides of vacuolar proteins, such as theN-terminal propeptide (NTPP) of sweet potato sporamin and the C-terminalpropeptide (CTPP) of tobacco chitinase can be used to target expressionof a protein to the vacuole.

By facilitating the transport of the protein into compartments insidethe cell, these transit peptides may increase the accumulation of geneproduct by protection from proteolytic degradation.

Production and Characterization of Stably Transformed Plants

Plant species may for instance be transformed by the DNA-mediatedtransformation of plant cell protoplasts and subsequent regeneration ofthe plant from the transformed protoplasts in accordance with procedureswell known in the art.

Any plant tissue capable of subsequent clonal propagation, whether byorganogenesis or embryogenesis, may be transformed with a vector of thepresent invention. The term “organogenesis”, as used herein, means aprocess by which shoots and roots are developed sequentially frommeristematic centers; the term “embryogenesis”, as used herein, means aprocess by which shoots and roots develop together in a concertedfashion (not sequentially), whether from somatic cells or gametes. Theparticular tissue chosen will vary depending on the clonal propagationsystems available for, and best suited to, the particular species beingtransformed. Exemplary tissue targets include leaf disks, pollen,embryos, cotyledons, hypocotyls, megagametophytes, callus tissue,existing meristematic tissue (e. g., apical meristems, axillary buds,and root meristems), and induced meristem tissue (e. g., cotyledonmeristem and ultilane meristem).

Plants of the present invention may take a variety of forms. The plantsmay be chimeras of transformed cells and non-transformed cells; theplants may be clonal transformants (e. g., all cells transformed tocontain the expression cassette); the plants may comprise grafts oftransformed and untransformed tissues (e. g., a transformed root stockgrafted to an untransformed scion in citrus species). The transformedplants may be propagated by a variety of means, such as by clonalpropagation or classical breeding techniques. For example, firstgeneration (or T1) transformed plants may be selfed to give homozygoussecond generation (or T2) transformed plants, and the T2 plants furtherpropagated through classical breeding techniques. A dominant selectablemarker (such as npt II) can be associated with the expression cassetteto assist in breeding.

Thus, the present invention provides a transformed (transgenic) plantcell, in planta or ex planta, including a transformed plastid or otherorganelle, e. g., nucleus, mitochondria or chloroplast.

Transformation of plants can be undertaken with a single DNA molecule ormultiple DNA molecules (i. e., co-transformation), and both thesetechniques are suitable for use with the expression cassettes of thepresent invention. Numerous transformation vectors are available forplant transformation, and the expression cassettes of this invention canbe used in conjunction with any such vectors. The selection of vectorwill depend upon the preferred transformation technique and the targetspecies for transformation.

Suitable methods of transforming plant cells include, but are notlimited to, microinjection (Crossway et al., 1986), electroporation(Riggs et al., 1986), Agrobacterium-mediated transformation (Hinchee etal., 1988), direct gene transfer (Paszkowski et al., 1984), andballistic particle acceleration using devices available from Agracetus,Inc., Madison, Wis. And BioRad, Hercules, Calif. (see, for example,Sanford et al., U.S. Pat. No. 4,945,050; and McCabe et al., 1988). Alsosee, Weissinger et al., 1988; Sanford et al., 1987 (onion); Christou etal., 1988 (soybean); McCabe et al., 1988 (soybean); Datta et al., 1990(rice); Klein et al., 1988 (maize); Klein et al., 1988 (maize); Klein etal., 1988 (maize); Fromm et al., 1990 (maize); and Gordon- Kamm et al.,1990 (maize); Svab et al., 1990 (tobacco chloroplast); Koziel et al.,1993 (maize); Shimamoto et al., 1989 (rice); Christou et al., 1991(rice); European Patent Application EP 0 332 581 (orchardgrass and otherPooideae); Vasil et al., 1993 (wheat); Weeks et al., 1993 (wheat). Inone embodiment, the protoplast transformation method for maize isemployed (European Patent Application EP 0 292 435, U.S. Pat. No.5,350,689).

It is particularly preferred to use the binary type vectors of Ti and Riplasmids of Agrobacterium spp. Ti-derived vectors transform a widevariety of higher plants, including monocotyledonous and dicotyledonousplants, such as soybean, cotton, rape, tobacco, and rice (Pacciotti etal., 1985: Byrne et al., 1987; Sukhapinda et al., 1987; Park et al.,1985: Hiei et al., 1994). The use of T-DNA to transform plant cells hasreceived extensive study and is amply described (EP 120516; Hoekema,1985; Knauf, et al., 1983; and An et al., 1985). For introduction intoplants, the chimeric genes of the invention can be inserted into binaryvectors as described in the examples.

Other transformation methods are available to those skilled in the art,such as direct uptake of foreign DNA constructs (see EP 0295959),techniques of electroporation (Fromm et al., 1986) or high velocityballistic bombardment with metal particles coated with the nucleic acidconstructs (Kline et al., 1987, and U.S. Pat. No. 4,945,050). Oncetransformed, the cells can be regenerated by those skilled in the art.Of particular relevance are the methods to transform foreign genes intocommercially important crops, such as rapeseed (De Block et al., 1989),sunflower (Everett et al., 1987), soybean (McCabe et al., 1988; Hincheeet al., 1988; Chee et al., 1989; Christou et al., 1989; EP 301749), rice(Hiei et al., 1994), and corn (Gordon Kamm et al., 1990; Fromm et al.,1990).

Those skilled in the art will appreciate that the choice of method mightdepend on the type of plant, i. e., monocotyledonous or dicotyledonous.

Agrobacterium tumefaciens cells containing a vector comprising anexpression cassette of the present invention, wherein the vectorcomprises a Ti plasmid, are useful in methods of making transformedplants. Plant cells are infected with an Agrobacterium tumefaciens asdescribed above to produce a transformed plant cell, and then a plant isregenerated from the transformed plant cell. Numerous Agrobacteriumvector systems useful in carrying out the present invention are known.These typically carry at least one T-DNA border sequence and includevectors such as pBIN19 (Bevan, 1984).

Methods using either a form of direct gene transfer orAgrobacterium-mediated transfer usually, but not necessarily, areundertaken with a selectable marker which may provide resistance to anantibiotic (e. g., kanamycin, hygromycin or methotrexate) or a herbicide(e. g., phosphinothricin). The choice of selectable marker for planttransformation is not, however, critical to the invention.

General methods of culturing plant tissues are provided for example byMaki et al., (1993); and by Phillips et al. (1988).

After transformation the transgenic plant cells are placed in anappropriate selective medium for selection of transgenic cells which arethen grown to callus. Shoots are grown from callus and plantletsgenerated from the shoot by growing in rooting medium. The particularmarker used will allow for selection of transformed cells as compared tocells lacking the DNA which has been introduced.

To confirm the presence of the transgenes in transgenic cells andplants, a variety of assays may be performed. Such assays include, forexample, “molecular biological” assays well known to those of skill inthe art, such as Southern and Northern blotting, in situ hybridizationand nucleic acid-based amplification methods such as PCR or RT-PCR;“biochemical” assays, such as detecting the presence of a proteinproduct, e.g., by immunological means (ELISAs and Western blots) or byenzymatic function.

The invention further provides a transgenic plant transformed with anucleotide sequence coding for a bZIP19 or a bZIP23 protein or both.Additionally, said plant may be transgenic for a protein selected fromthe group consisting of heavy metal transporters, preferably HMA2, HMA3or HMA4, YSL proteins, preferably YSL, preferably YSL1 or YSL3, ZIP orIRT proteins, ZIF proteins, NAS proteins, MRP proteins, FRD3 and MTPs.

As will be shown in the Examples, the transgenic plants according to theinvention are tolerant to Zn deficiency, i.e. they are able to adapt toenvironmental situations in which there is no or hardly no zinc in thesubstrate on which the plant is growing. Further advantage of thetransgenic plants of the current invention is that they are able toaccumulate Zn. This is not only an advantage for bioremediation, but incases of plants where Zn is stored in edible parts (such as leaves,roots, beets, berries or tubers) the plants will be suitable for dietsin areas where the Zn concentration in the food is insufficient in thenormal diet. This use is also known under the name of‘biofortification’. Normally the Zn in the plant will be lessbioavailable because part of it is complexed with phytate orpolyphenols. Thus preferably the plants used in this respect are plantsthat comprise low levels of phyate or and/or polyphenoles.

On the other hand, the transgenic plants of the invention are veryuseful in circumstances where there is an excess of zinc in thesubstrate. The transgenic plants of the inventions are capable of takingup the metal and storing it in their tissues and/or vacuole. In thisway, the plants can be used as sanitation plants to extract zinc fromthe soil. The metal thus will be concentrated in the plant biomass,which can be harvested and disposed off in a convenient way. Thismethod, known as phytoremediation, is especially useful if large areasof soil are contaminated with zinc (such as is the case in theneighbourhood of zinc mines). In such a case, phytoremediation is acommercially very attractive way of cleaning the surroundings fromexcess metal.

A further part of the invention is the use of a bZIP19 and/or a bZIP23protein or an ortholog thereof, or a nucleotide sequence encosing forsuch a protein or ortholog, in the methods of the invention describedabove, and/or for making a plant as described above and progeny thereof.

The following enabling Examples serve to further illustrate theinvention, and are not intended to define limitations or restrict thescope of the subject invention.

EXAMPLES Example 1—Material and Methods Plant Growth

Arabidopsis ecotype Columbia (Col-0) was used in all experiments. Plantswere grown in climate chambers with 16h light at 22° C., 8 h at 20° C.,120 μmol photons m⁻² s⁻¹ and 50% relative humidity. Previous togermination, seeds had a 3-day stratification treatment in a cold roomat 4° C. in the dark to promote uniform germination. For geneticanalysis and transformation, plants were grown in pots with peat. Forthe plate-based assay, seeds were surface-sterilized using vapour-phaseseed sterilization and sown on plates with MS media (Duchefa Biochemie,Haarlem, The Netherlands) supplemented with 1% sucrose and adjusted topH 5.8. The MS media was prepared either without zinc (Zn−), with 30 μMZnSO₄ (Zn+) or with 300 μM ZnSO₄ (Zn++). For the hydroponically grownplants, seeds were sown on 0.55% agar-filled tubes and grown on amodified half-strength Hoagland's nutrient solution prepared with either0.05 μM (Zn−), 2 μM ZnSO₄ (Zn+), or 25 μM ZnSO₄ (Zn++). The hydroponicsystem consisted of 8-liter-capacity containers (46×31×8 cm), with anon-translucent 3-mm thick plastic lid containing holes for placing 9×5agar-filled tubes. The nutrient solution was replaced once in the firstweek and twice in the weeks thereafter.

Yeast Complementation Experiment

A 1.3-kb full-length cDNA clone corresponding to AtZIP4 (APD09D10R,genebank accession number AV524735) was kindly provided by Kazuza DNAResearch Institute (Kisarazu, Chiba, Japan). The open-reading frame ofZIP4 was amplified with proofreading DNA polymerase (Pfu native;Stratagene, La Jolla, Calif., USA), with PCR conditions as recommendedby the manufacturer, and using the primers

(SEQ ID NO: 5) 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAACTCTTGTTCCCATGA TC-3′and (SEQ ID NO: 6) 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTATATTATTTGATTCTACAG-3′containing Gateway recombination sites (underlined). The fragment wascloned into the entry vector pDONR207 (Invitrogen) by in vitrosite-directed recombination for further recombination into the yeastexpression vector pFL613 (20). The ATG start-codon of the cloned ZIP4cDNA was in frame with the pFL613 ATG start-codon and withoutstop-codons upstream. The construct was verified by sequencing.

A Saccharomyces cerevisiae zrt1zrt2 mutant, ZHY3, and its parentalwild-type strain DY1457 were used (6). Yeast cells were grown onsynthetic defined liquid media (SD) supplemented with auxotrophicrequirements and 2% (w/v) glucose. Both yeast strains were transformedwith the pFL613 empty vector, and zrt1zrt2 was transformed with pFL613containing AtZIP4, using a standard yeast transformation procedure (21).zrt1zrt2 complementation was tested by a drop spotting assay, spottingdiluted cultures from a single colony of each transformant on selectiveSD-URA agar plates. The media was made zinc-limiting by adding EDTA (1mM) and citrate (pH 4.2) (22) and supplemented with 0.4 (zinc-limitingmedia) or 0.8 mM ZnCl₂. Five colonies of each transformant were tested.zrt1zrt2 complementation was also tested by measuring the OD₆₀₀ in 5-mlcultures of the described SD-URA media with 0.4 mM ZnCl₂ inoculated witha 60 μl (0.8 mM ZnCl₂) single-colony overnight pre-culture at OD₆₀₀=1.Three independent experiments were performed.

Construction of Reporter Vectors for the Yeast One-Hybrid

The bait sequence in six reporter vectors (named A to F) werePCR-amplified fragments of the ZIP4 promoter. These fragments coveredthe full promoter, starting −1049 bp upstream of the start codon and hadan overlap between fragments of 60 to 80 bp. The fragments wereamplified from Arabidopsis genomic DNA using proofreading polymerase(Pfu native; Stratagene, La Jolla, Calif., USA), with PCR conditions asrecommended by the manufacturer, and using primers with 5′-overhangscompatible with EcoRI/SacI (table 1). The fragments were intermediatelycloned into the pCR-Blunt II-TOPO vector (Invitrogen) according to themanufacturer's recommendations. The TOPO vector with each of the baitswas digested with EcoRI/SacI and the bait fragment was extracted fromagarose gel (Qiagen Gel Extraction kit). The bait of the reporter vectorG consited of a trimer of the following motif: ATGTCGACAT/C. Twoantiparallel oligonucleotides, one representing the sense strand and theother its antisense complement strand, and containing 5′-overhangscompatible with EcoRI/SacI, were synthesized (table 1).

TABLE 1Forward and reverse primers used to amplify bait fragments A-F from the ZIP4promoter, and sense and antisense complement strands used to synthesise thethree-tandem repeat of the motif A TGTCGACAT/C(G), to be used as baitfragment G. SEQ ID Fragment Primer sequence NO: A Forward5′-GAATTCAAGCTTTGGAAAGTGAAGTGGA-3′  8 Reverse5′-GAGCTCCAATTTCAAACCAGTA-3′  9 B Forward5′-GAATTCTGTATATCTGATCTTCTCTGCTG-3′ 10 Reverse5′-GAGCTCAAGCTAAAAGGACGGTAACT-3′ 11 C Forward5′-GAATTCTTCATCCTATTGCTTGG-3′ 12 Reverse 5′-GAGCTCATTTTCCCATTTGTTCCAC-3′13 D Forward 5′-GAATTCTCTGCAGTAGACTTGAC-3′ 14 Reverse5′-GAGCTCCCCAATCTTGTCTAT-3′ 15 E Forward5′-ATCGGAATTCGTGAGAAAACAGAATAACGC-3′ 16 Reverse5′-GAGCTCCCATGGGAACAAGAGTTTAT-3′ 17 F Forward5′-ATCGGAATTCGTGAGAAAACAGAATAACGC-3′ 18 Reverse5′-CGTAGAGCTCTGGAGAAAGAGTGAAAGAGT-3′ 19 G Forward5′-AATTCATGTCGACATATGTCGACATATGTCGACACGAGCT-3′ 20 Reverse5′-CGTGTCGACATATGTCGACATATGTCGACATG-3′ 21

0.1 μg of each oligonucleotide strand were mixed in 10 μl of 50 mM NaCl,annealed by heating at 70° C. for 5 min, and slowly cooled down to roomtemperature. The digested, PCR-derived baits (A to F) and the annealedoligonucleotide (G), were cloned into pHISi, previously digested withEcoRI/SacI according to the manufacturer's recommendations. Eachreporter vector was confirmed by digestion analysis and sequencing.

Yeast One-hybrid Screening

The cDNA expression library was constructed with mRNA from Arabidopsisinflorescence obtained using an mRNA purification kit (AmershamBioscience). Subsequently, a Gateway compatible cDNA entry library wasconstructed making use of the CloneMiner cDNA library Construction Kit(Invitrogen, Carlsbad). This cDNA entry library had a titer of 5×10⁷ cfuml⁻¹ and it was transferred into the pDEST22 vector (Invitrogen) via anLR recombination reaction following the protocol provided by themanufacturer, yielding an expression library with a titer of 2×10⁶ cfum1⁻¹. The reporter vectors were introduced into yeast strain PJ69-4A(James, P. et al., 1996, Genetics 144:1425-1436). For this purpose,yeast cells were transformed with digested (Xho1) linearized pHISireporter vector using a standard yeast transformation procedure (Gietz,R. D. et al., 2002, Meth. Enzymol. 350:87-96). The empty pHISi vector,digested and undigested, and a non-integrative reporter vector were usedas controls. The cDNA expression library screening was performedfollowing the Large-Scale Yeast Transformation Protocol (PT3024-1;Clontech) which yielded a transformation efficiency of 5 to 9×10⁵ cfuμg⁻¹ DNA. Screening with all the reporter strains was performed onmedium lacking His and in the presence of 20-40 mM 3-aminotriazole (3AT,optimal concentration was optimized for each reporter strain). Therewere in total 18 positive interactions (7 with bZIP19 and 11 withbZIP23) when using the reporter vectors E, F and G (FIG. 2A). Positivecolonies were selected and the cDNA clone of the GAL4-AD library vectorwas isolated and sequenced.

Identification of T-DNA Insertion Mutants

T-DNA SALK lines were obtained from the Nottingham Arabidopsis StockCenter (NASC). The T-DNA was inserted 18 bp upstream of the bZIP19(At4g35040) start codon in m19 (salk_144252), and 91 bp upstream of thebZIP23 (At2g16770) start codon in m23 (salk_045200). The T-DNA insertionevents were confirmed by PCR analysis with gene-specific primers of LPand RP described by the Salk Institute Genomic Analysis Laboratory and aT-DNA border primer LBa1. Homozygous plants for each T-DNA insertionwere selected. In order to obtain a double T-DNA insertion mutant,m19m23, the progeny of a cross between m19 and m23 plants were selectedby PCR analysis for homozygosity of each T-DNA insert.

Quantitative RT-PCR Analysis

Seedlings of Arabidopsis wild-type (wt), and T-DNA insertion mutantsm19, m23 and m19m23 grown for three weeks in MS medium at either Zn−,Zn+or Zn++ conditions were harvested. Seedlings from a single plate, pergenotype and per treatment were pooled (6 to 8 seedlings) andhomogenized in liquid nitrogen. For each genotype and treatment,seedlings were harvested from 3-4 different plates, representingindependent experiments. Total RNA of the seedlings was extracted withan RNAeasy plant RNA kit (Qiagen) and treated with DNAse to eliminateany genomic DNA (Fermentas). The kits were used according tomanufacture's instructions. First-strand cDNA was synthesized from 1 μgof total RNA using the iScript™ cDNA Synthesis Kit (Bio-Rad).Gene-specific primers for quantitative RT-PCR were designed according todatabase genome sequence information for Arabidopsis and using VectorNTI software (Invitrogen) (table 2).

TABLE 2 Forward and reverse primers used in the quantitative RT-PCR todetermine transcript expression of ZIP4, ZIP1, ZIP3, ZIP5,ZIP9, ZIP12, IRT3, ZIP2, bZIP19, bZIP23 and bZIP24 genes. SEQ IDFragment Primer sequence NO: ZIP4 Forward 5′-GATCTTCGTCGATGTTCTTTGG-3′22 Reverse 5′-TGAGAGGTATGGCTACACCAGCAGC-3′ 23 ZIP1 Forward5′-GGACACACACATGGTTCGAC-3′ 24 Reverse 5′-GATAGTGCAGCCATGAGTGG-3′ 25 ZIP3Forward 5′-CAGAAACATGTTTCTTCTTCGTCAC-3′ 26 Reverse5′-CGCAATAAATCCGGTGAACG-3′ 27 ZIP5 Forward 5′-CGGGATTGTTGGCGTGGAAT-3′ 28Reverse 5′-CCAAGACCCTCGAAGCATTG-3′ 29 ZIP9 Forward5′-CAATAATCATAGGAATATCGCTTGG-3′ 30 Reverse 5′-AGAAAGCCATCATGGCAGAT-3′ 31ZIP12 Forward 5′-CAATGTTGATTGAATCCTTTGC-3′ 32 Reverse5′-CCATGAGAATGTCCTTGTGA-3′ 33 IRT3 Forward 5′-ATATGTTGGCGGGTGGCACG-3′ 34Reverse 5′-GCTTCCCTCTCTTGCTTCCG-3′ 35 ZIP2 Forward5′-TAATAACAACCACGTCGGAG-3′ 36 Reverse 5′-AGCAAAGCTGTGTCTCCAAA-3′ 37bZIP19 Forward 5′-TTCTCCCGGATGAGAGCGATGA-3′ 38 Reverse5′-GCTGATTCACCGCCCTAAGCCT-3′ 39 bZIP23 Forward5′-TAATCAGCTGTTGAAGAGGT-3′ 40 Reverse 5′-TCATGTATGAGTAAGGCACG-3′ 41bZIP24 Forward 5′-TCTCAGGATCAGCAAGAGAA-3′ 42 Reverse5′-TCAGTTTCCACCATTTCTTGG-3′ 43

Amplicon lengths were between 150 and 240 bp and all primer combinationshad at least 85% efficiency. The absence of genomic DNA was confirmed byperforming a no-amplification control (without reverse transcriptasereaction) for every sample. For the PCR reaction, 5 μl of a 100×dilution of the cDNAs, corresponding approximately to 2.5 ng of RNA,were used as template. In addition, the reaction contained 12.5 μl iQ™SYBR® Green Supermix (Bio-Rad) and 5 pmol of forward and reverse primers(Invitrogen) in a total volume of 25 μl. The PCR reactions wereperformed in a 96-well plate with an iCycler thermal cycler and aniCycler iQ Real Time PCR System (Bio-Rad). The following standardthermal profile was used: 3 min at 95.0° C., followed by 40 cycles of 15sec at 95.0° C. and 1 min at 60.0° C. 18S rRNA was used as an internalcontrol, to normalize the amount of template cDNA. Reactions wereperformed in 3-4 biological replicas and 2-6 technical replicas perbiological replica for each genotype x treatment. Relative transcriptlevels (RTL) were calculated with the 2^(−ΔΔCT) method (24).

Determination of Zinc Concentration

Roots and shoots of four-week-old hydroponically grown Arabidopsiswild-type (wt), T-DNA insertion mutants m19, m23 and m19m23, wereharvested, consisting of three plants per genotype x three treatments(Zn−, Zn+, Zn++) x two independent experiments. The root systems weredesorbed with ice-cold 5 mM PbNO₃ for 30 min. Roots and shoots wereanalyzed for zinc concentration using flame atomic absorptionspectrometry (Perkin Elmer 1100B).

Generation of Constructs

To generate overexpressor constructs for transformation of theArabidopsis double T-DNA insertion mutant (m19m23), full-length cDNAs ofAtbZIP19 and AtbZIP23 (clones GSLTSIL54ZHO9 and GSLTFB35ZE06,respectively) were obtained from the CNRGV (Centre National deRessources Génomiques Végétales, France) in pCMV SPORT6 cloning vector,containing Gateway recombination sites. The cDNAs were cloned into theentry vector pDONR207 (Invitrogen) by in vitro site-directedrecombination, for further recombination into the overexpressor vectorpGD625 (De Folter, S. et al., 2005, Plant Cell 17:1424-1433). Theconstructs pCaMV35S::bZ1P19 (OX19) and pCaMV35S::bZ1P23 (0X23) wereverified by digestion analysis and sequencing and transformed byelectroporation into Agrobacterium tumefaciens strain AGLO.Subsequently, m19m23 and Arabidopsis wild-type plants were transformedby floral dipping (Clough, S. J. et al., 1998, Plant J. 16:735-743).Independent transformed lines were selected for a single insertion locusby antibiotic resistance and 3:1 segregation ratio of T2 seedlings. Theoverexpression of bZIP19 or bZIP23 was confirmed by RT-PCR. Five,respectively three independent lines of m19m23-OX19 and m19m23-0X23 wereanalysed with 10 seedlings per line, grown in MS medium at either Zn− orZn+ conditions, in two replicate plates per line. Arabidopsis wild-typeand m19m23 plants were used as controls.

Micro-Array Analysis

Arabidopsis wild-type (wt) and T-DNA insertion double mutant m19m23plants were grown in hydroponics medium as described above. They weregrown for three weeks with normal zinc supply (Zn+, 2 μM ZnSO₄) and oneweek with low zinc supply (Zn−, 0.05 μM ZnSO₄) or normal zinc supply(Zn+). Roots of four plants per genotype and per treatment (Zn−/Zn+)were pooled in a two biological replica experiment, and RNA wasextracted with the RNAeasy plant RNA kit (Qiagen). Transcriptomes wereanalysed using 1 μg of total RNA as starting material. Targets wereprepared with the one-cycle cDNA synthesis kit followed bybiotin-labelling with the IVT labelling kit (GeneChip One-cycle targetlabelling and control reagents, Affymetrix, High Wycombe, U.K.) andhybridized to ATH1 gene chip for 16 h as recommended by the supplier(Gene expression analysis manual, Affymetrix). Raw data files wereprocessed and quantile normalized in Bioconductor/R (27). Differentialexpression of each gene was tested for by applying an Empirical Bayesregularized t-test (Smyth, G. K., 2004, Stat. Appl. Genet. Mol. Biol. 3,Iss. 1, Article 3)). The p-values were corrected for multiple testingusing the approach of Benjamini and Hochberg (1995, J. Royal Stat., Soc.B 57:289-300), providing control of the false discovery rate (FDR).

Statistics

Data analysis and statistics were done using Microsoft Excel and SPSS15.0 for Windows. Statistical analysis of zinc concentration and dryweight data, from Arabidopsis and mutant lines, was performed by one-wayANOVA followed by a post-hoc Tukey test.

Results

In Arabidopsis, several members of the ZIP family of metal transportersmainly involved in cellular uptake, are transcriptionally induced inresponse to zinc deficient conditions and thought to constitute themajor gateway of zinc into the plant. The ZIP4 gene in particular isstrongly induced in roots upon zinc deficiency (FIG. 3A). We used it tocomplement the increased zinc requirement of the Saccharomycescerevisiae zrt1zrt2 mutant defective in high- and low-affinity zincuptake (Zhao, H. et al., 1996, Proc. Natl. Acad. Sci. USA93:2454-2458)), and showed it indeed encodes a zinc transporter (FIG. 4,B and C). We cloned the promoter of the ZIP4 gene upstream of the GUSreporter gene and expressed this construct in Arabidopsis. GUSexpression was apparent only when plants were grown on zinc deficientmedium, analogous to endogenous ZIP4 gene expression (data not shown).To identify transcription factors controlling ZIP4 expression, we usedsix overlapping ZIP4 promoter fragments as baits in a yeast-one-hybridassay. We also used three tandem repeats of a 10-bp palindrome motif,present in two copies close to the predicted ZIP4 transcription start,as additional bait (FIG. 2A; fragments A to G). Several clones of twocDNAs were identified, but only when screening with baits containing twoor three copies of the 10-bp palindrome (FIG. 2A; fragments E, F and G).These cDNAs corresponded to bZIP19 (At4g35040, NM_119670.4) and bZIP23(At2g16770, NM_119670.4), two genes of the basic region/leucine zippermotif (bZIP) family of transcription factors. Arabidopsis contains 75members of the bZIP family (Jakoby, M. et al., 2002, Trends Plant Sci.7:106-111).

To investigate the involvement of these three bZIP genes in controllingadaptation of plants to low zinc supply, we analysed transcript levelsby quantitative RT-PCR (qPCR) on three-week-old wild-type seedlingsgrown on agar plates at three different zinc concentrations. Expressionof bZIP19 and bZIP23 genes decreased to about half upon increased zincsupply, with bZIP19 slightly higher expressed than bZIP23 (FIG. 2B).bZIP24 had lower transcript levels than the other two, and expressionwas not obviously affected by the zinc status of the medium, thereforewe concluded that bZIP19 and bZIP23 are involved in the response to zincdeficiency in plants.

To determine their mutant phenotypes, we obtained homozygous T-DNAinsertion lines for the bZIP19 and bZIP23 genes (respectively named m19and m23) which were devoid of full-length bZIP19 or bZIP23 transcript(FIG. 5, A and B). We crossed both single mutant lines to generate adouble mutant line (m19m23). Wild type plants and single or doublemutants grown on soil did not show any obvious phenotypic differences(data not shown), however when grown on zinc-deficient agar media (Zn−),only the double mutant line was hypersensitive to zinc-deficiency (FIG.4A). Three-week-old seedlings showed very poor growth and strongchlorosis. To exclude in vitro effects during tissue culture, we grewplants for longer time on hydroponics medium, where they could developnormally. Four-week-old double mutant plants, growing at low zinc supply(Zn−) showed a strong growth reduction compared to wild-type plants, asdetermined by dry weight comparison (FIG. 4, B and C). These plants alsohad a decreased zinc uptake, with only 58% and 73% of the respectiveshoot and root zinc concentration of wild-type plants (FIG. 4C). At lowzinc supply on hydroponics, also the m19 single mutant showed reductionin growth and decreased zinc uptake when compared to wild type (FIG.4B). When growing in zinc-sufficient (Zn+) or zinc-excess media (Zn++),we did not see differences between the mutants and the wild-type (FIG.4, A and B), neither for zinc content nor for dry weight (FIG. 6,7). Toprove that mutations in bZIP19 and bZIP23 indeed caused the phenotype ofthe m19m23 double mutant we expressed either the bZIP19 or the bZIP23cDNA, each under control of the CaMV 35S promoter, in the double mutant.This fully complemented the zinc deficiency hypersensitive phenotype(FIG. 8A).

These findings show that bZIP19 and bZIP23 encode essentialtranscription factors that control the zinc deficiency response inplants. These genes act redundantly, with bZIP19 only being partiallyredundant. bZIP transcription factors generally act as dimers. Theirredundancy suggests that bZIP19 and bZIP23 act as homo(di)mers, in linewith previous predictions (Deppmann, C.D. et al., 2006, Mol. Biol. Evol.23:1480-1492). The DNA target sequence for binding bZIP19 and bZIP23should be within the 10-bp imperfect palindrome present in two copies inthe ZIP4 promoter, as three tandem copies of this sequence weresufficient to identify both bZIPs in the yeast-one-hybrid assay. Wetherefore called the palindrome consensus sequence (RTGTCGACAY) (SEQ IDNO:44) a Zinc Deficiency Response Element (ZDRE). The ZDRE does not havethe typical ACTG core as found in the A-box (TACGTA), C-box (GACGTC) orG-box (CACGTG) DNA elements, to which plant bZIPs are known topreferentially bind. Although also other binding sites have beenreported (Choi, H. et al., 2000, J. Biol. Chem. 275:1723-1730; Fukazawa,I. Et al., 2000, Plant Cell 12:901-915) the ZDRE is not among them.

To further confirm that the ZDRE is indeed the characteristic ciselement to target genes for transcriptional control by bZIP19/23, wescreened the promoters of other ZIP transporter genes for ZDREs. Of the15 Arabidopsis ZIP genes (Maser, P. et al., 2001, Plant Physiol.126:1646-1667), approximately half are described to be transcriptionallyinduced under zinc deficiency conditions compared to normal zinc supply(Grotz, N. et al., 1998, Proc. Natl. Acad. Sci USA 95:7220-7224; Van deMortel, J. E. et al., 2006, Plant Physiol. 142:1127-1147; Wintz, H. etal., 2003, J. Biol. Chem. 278:47644-47653). Only ZIP1, ZIP2, ZIP3, ZIP4and IRT3 encode proteins characterized as zinc transporters (Grotz,supra; Lin, Y.-F. et al., 2009, New Phytol. 182:392-404) although othersare likely to transport zinc too. ZIP1, ZIP3, ZIP4, ZIP5, ZIP9, ZIP12and IRT3 contain one or two ZDRE copies in their promoters and thesegenes do not show the typical induction of expression in the m19m23double mutant under zinc-deficient conditions as we saw in the wild-type(FIG. 8B). Expression of ZIP2, which is not zinc-deficiency induced andhas no ZDRE sequence in its promoter, is not affected when comparingmutant and wild-type (FIG. 8B). In order to determine the effect of lossof bZIP19/23 function on global gene expression we performed amicro-array experiment comparing roots of hydroponically grownfour-week-old m19m23 double mutant plants with those of wild-typeplants, treated in their last week with low zinc supply (Zn−). Only 23genes showed a statistically significant alteration of transcript levelsexceeding a 1.5-fold difference threshold (Table 3).

Among those, 16 were down-regulated in the double mutant, includingbZIP19 (a probe for bZIP23 was not included in the micro-array used). Ofthe 15 remaining genes, 11 are known to be induced in wild typeArabidopsis roots upon zinc deficiency (Van de Mortel et al., supra) andnine contain one or more copies of ZDRE in their promoter regions. Wethink these are the direct targets of bZIP19 and bZIP23, important forthe primary zinc deficiency response, and the other genes represent asecondary effect. These findings confirm the important role of the ZDREand the bZIP19/23 genes in controlling Arabidopsis zinc deficiencyresponse. It also shows that the strong negative effect on growth and onzinc concentration of the m19m23 mutant when grown under zinc deficiency(FIGS. 4, A, B and C) is largely explained by reduction in expression ofa relatively small group of zinc homeostasis genes involved in uptakeand translocation of metals.

TABLE 3Differentially expressed genes detected by comparative micro-array analysis ofthe root transcriptome of Arabidopsis wild-type and m19m23 double mutant plants.Roots of four-week old plants grown in hydroponics exposed in their last week to zincdeficiency (Zn-) were used. Fold change (FC) ≥1.5 and adjusted p-values (Benjamini-Hochberg, BH) ≥0.05 were used as cut-offs. Average expression value (log2 scale)of the gene model in the data set is indicated as A. Adjusted Gene FCp-value Annotation (www.arabidopsis.org) Model (1.5) A (BH)down-regulated bZIP transcription factor family protein AT4G35040 -35.87 7.31 9.27E-08 ZIP3 (ZINC TRANSPORTER 3 PRECURSOR)* AT2G32270 -26.23 9.67 1.35E-09 phosphatidylinositol 3- and 4-kinase family protein/AT5G24240  -8.43  8.20 3.57E-08 ubiquitin family proteinZIP5 (ZINC TRANSPORTER 5 PRECURSOR)* AT1G05300  -8.34  6.90 4.15E-07ZIP4 (ZINC TRANSPORTER 4 PRECURSOR)* AT1G10970  -7.19  7.68 9.27E-08ZIP9 (ZINC TRANSPORTER 9 PRECURSOR)* AT4G33020  -3.23  6.90 4.41E-04ZIP1 (ZINC TRANSPORTER 1 PRECURSOR)* AT3G12750  -3.21  7.33 1.10E-05nicotianamine synthase, putative* AT5G56080  -3.02  9.24 1.28E-02ATPAP27/PAP27 (purple acid phosphatase 27) AT5G50400  -2.51  9.791.10E-05 nicotianamine synthase, putative* AT1G56430  -2.46  7.681.98E-03 ATARP9 (ACTIN-RELATED PROTEIN 9) AT5G43500  -2.33  7.283.16E-05 FRD3 (FERRIC REDUCTASE DEFECTIVE 3) AT3G08040  -1.78  8.611.98E-03 prolyl oligopeptidase, putative/prolyl endopeptidase, AT1G20380 -1.77  8.28 6.20E-03 putative/post-proline cleaving enzyme, putative*WR3 (WOUND-RESPONSIVE 3) AT5G50200  -1.60 11.63 2.48E-02ZIP10 (ZINC TRANSPORTER 10 PRECURSOR)* AT1G31260  -1.60  6.30 3.09E-02similar to unknown protein [Arabidopsis thaliana] AT4G04990  -1.59  8.414.22E-03 (TAIR:AT1G61260.1) up-regulated LAC2 (laccase 2) AT2G29130  1.82  7.01 1.82E-02 ANR1; DNA binding/transcription factor AT2G14210  1.71  8.90 2.85E-02 similar to unknown protein [Arabidopsis thaliana]AT1G21680   1.57  8.98 2.85E-02 (TAIR:AT1G21670.1)ATEBP/ERF72/RAP2.3 (RELATED TO AP23) AT3G16770   1.55  9.68 4.18E-02COBL2 (COBRA-LIKE PROTEIN 2 PRECURSOR) AT3G29810   1.54  7.40 3.42E-02kelch repeat-containing F-box family protein AT1G80440   1.52 11.059.48E-03 ATPSK2 (PHYTOSULFOKINE 2 PRECURSOR) AT2G22860   1.52  9.204.24E-02 *Indicates genes that contain one or more copies of the ZDRE intheir promoter region.

In summary, the bZIP19 and bZIP23 transcription factor function isessential for the response and adaptation of plants to low zinc supply.The identification of these transcription factors, as well as the ZDREelement they bind to and the target genes they regulate, constitutes animportant step forward towards a full understanding of zinc homeostasisin plants.

Example 2

Arabidopsis plants, accession Col, were transformed with OX19 or OX23(for the construction of these vectors, see Example 1) and homozygous T3plants were obtained. Three high expressing lines containing either OX19(genotypes 14, 15, 19) or OX23 (genotypes 16, 17, 18) were selected.Seeds of these lines were germinated and grown in two replicates onhydroponic medium containing no additional Zn (0 μM Zn; Zn deficiency),2 μM Zn (normal Zn), or 25 μM Zn (excess Zn). Plants were photographedafter 6 weeks and sampled for biomass analysis.

When comparing plants grown at normal Zn conditions, there was noobvious difference in visible phenotype (data not shown). However, whenplants grown on Zn deficient medium were compared, lines 19 (OX 19), 16and 17 (OX23) were generally larger and greener (FIG. 9). When examinedfor biomass production, indeed all lines were found to produce morebiomass than WT, although at this small scale experiment, this was onlyfound to be significant for line #16 (FIG. 10A). When plants grown atnormal Zn supply were examined for biomass production, they weregenerally found to have less or equal biomass compared to WT (FIG. 10B),although the observed differences were not statistically significant.Zinc concentrations were determined in shoots of all lines grown atnormal zinc and zinc deficiency. The shoot Zn concentrations of plantsgrown at normal zinc were generally not different from WT (data notshown), however, at zinc deficiency, the concentration of zinc in alllines was lower than WT (FIG. 11), with especially lines 18 and 19showing only 60% of the Zn concentration of WT. This means that althoughplants are not able to acquire more Zn, due to depletion of Zn in themedium, some OX 19 and OX23 lines are able to produce more biomass withthe same amount of Zn, indicating they are more Zn deficiency tolerantand more Zn efficient.

Example 3

For this experiment, a new pZIP4::bZIP19 construct was made. Thiscontains the full Arabidopsis bZIP19 cDNA fused downstream of thepromoter of the zinc deficiency responsive Arabidopsis ZIP4 gene. Therationale of the experiments is to test if overexpression of bZIP19 orbZIP23 function will enhance the tolerance to Zn deficiency, compared towild type Arabidopsis plants. Overexpression of bZIP19 function in thisexperiment, in contrast with the experiment described in Example 2, ismediated by the promoter of the Zn deficiency transcriptionallyresponsive ZIP4 zinc transporter gene. Thus, overexpression of bZIP19function will be predominantly in the cells and tissues in whichnormally the Zn deficiency response is active, albeit expected at higherlevels than in WT plants.

Arabidopsis plants, accession Col, were transformed with thepZIP4::bZIP19 construct and homozygous T3 plants were obtained. In total6 lines were selected, containing the T-DNA inserted at one locus andshowing an undiminished hygromycin resistance phenotype after threesuccessive generations as evidence of stable transformation. WT andpZIP4::bZIP19 plants were grown in two replicates on medium with normalZn (2 μM Zn), or with no Zn added (0 μM Zn) to cause Zn deficiency.After four weeks of growth, photos were taken to determine anydifferences in visible phenotypes (FIG. 12). At this stage, plants arenot yet showing visible Zn deficiency symptoms, but show growthretardation, compared to plant grown on normal Zn (data not shown). Atthis stage, it is clear that WT plants in general have smaller rosettesthan pZIP4::bZ1P19 transformed plants.

1. A method to produce a modified plant, said method comprisingproviding said plant with nucleic acid encoding bZIP19 (SEQ ID NO: 1)operably linked to a promoter induced by zinc deficiency.
 2. The methodof claim 1, wherein said modified plant is grown under zinc deficiencyconditions and exhibits increased tolerance to Zn deficiency as comparedto an unmodified plant.
 3. The method of claim 1, which wherein saidmodified plant is grown under zinc deficiency or zinc sufficientconditions and has increased biomass as compared to an unmodified plant.4. The method of claim 1, wherein said modified plant is selected forincreased zinc in its shoots as compared to an unmodified plant.
 5. Themethod of claim 1, wherein said modified plant is further provided witha nucleic acid encoding bZIP23 (SEQ ID NO: 2).
 6. The method of claim 1,wherein said modified plant is additionally provided with one or morenucleic acids that encode a protein selected from the group consistingof heavy metal-associated (HMA), yellow stripe 1-like (YSL), zincregulated transporter IRT-like protein (ZIP), iron-regulated transporter(IRT), zinc induced facilitator (ZIF), nicotianamine synthase (NAS),multidrug resistance protein (MRP), ferric reductase defective 3 (FRD3)and metal tolerance proteins (MTPs).
 7. The method of claim 6 whereinthe the protein is selected from the group consisting of HMA2, HMA3,HMA4, YSL1, and YSL3.
 8. The method of claim 1, wherein said promoter isthe ZIP4 promoter.